Nisin Production Research Articles

Aeration and fermentation strategies on nisin production.

To optimize nisin production in Lactococcus lactis by using different aeration and fermentation strategies. The nisin titer and specific nisin production rate reached maximum values of 11,900IU/ml and 4110IU/g/h, respectively, in aerobic batch fermentation with glucose as C source. These values were higher than in anaerobic batch fermentation (10,700IU/ml and 3260IU/g/h, respectively). The maximum specific nisin production rates appeared earlier in aerobic batch fermentation, which suggests that nisin production is stimulated by aeration. Different fermentation strategies were compared: maximum nisin production (15,400IU/ml) was achieved with fed-batch fermentation with a variable rate of feeding under aerobic conditions. Nisin production can be stimulated by aeration, which goes against the typical conditions involving strict anaerobiosis.

Regulatory potential of post-translational modifications in bacteria.

Bacteria are often viewed as simple organisms, with very basic and robust cellular regulation, optimized for rapid growth. While they certainly fit that description, bacteria also possess an amazing capacity for adaptation, and diversity of survival strategies, including variations of cell morphology, size, mode of growth and developmental behavior. Post-translational modifications (PTMs) of proteins contribute significantly to bacterial adaptability and cell cycle control. Research on PTMs in bacteria started with the assumption that they lack many features regularly found in more complex organisms. However, ongoing investigation keeps revealing new types of PTMs of bacterial proteins. This research topic gathers a number of articles discussing the advanced methods for systematic study of bacterial PTMs, approaches to utilize PTMs for biotechnological purposes, and revealing new cellular functions controlled by PTMs. Most bacterial PTMs are dynamic and reversible. This allows the cell to exploit them as regulatory devices. It also means that the full understanding of the cellular roles of different PTMs necessitates global, quantitative and time-resolved studies. One seminal paper in this topic reports a novel proteomics approach for such quantitative studies: the Intensity Based Absolute Quantitation (iBAQ). Using this approach, the authors have quantified the expression and the occupancy of various PTM sites in the proteome of Escherichia coli (Soufi et al., 2015). The authors report remarkable differences in expression and occupancy of PTMs sites under different growth conditions. The dataset comprised 2300 proteins, which is close to 90% of the expressed proteome. This is an important landmark, since proteomics in general has not yet attained the same level of coverage that global transcriptome studies can achieve. Among different bacterial PTMs, protein phosphorylation is the most extensively studied one, and in this topic it features prominently. Several papers present global studies of protein phosphorylation in bacteria (Spat et al., 2015), including important bacterial pathogens (Fortuin et al., 2015; Nakedi et al., 2015). The focus on pathogens is understandable, since protein phosphorylation, and several other PTMs, are heavily involved in different infection strategies displayed by bacterial pathogens (Michard and Doublet, 2015). In addition to phosphoproteome studies, interactomics is also featured as a useful approach to chart the phosphorylation-based regulatory networks. It enables researchers to trace the connections among protein kinases, phosphatases, and their substrates (Shi et al., 2014a). This approach for reconstructing phosphorylation networks highlights the capacity of bacterial proteins kinases from different families to interact with, and phosphorylate, each other (Shi et al., 2014b). Protein-tyrosine kinases, and their various roles in bacterial physiology were in the focus of several review and opinion articles in this topic (Barak, 2014; Gerwig and Stulke, 2014; Mijakovic and Deutscher, 2015). Other topic contributions highlight the broad spectrum of PTMs involved in key cellular processes. In particular, novel modifications are discussed: redox regulation via reversible S-thiolation (Loi et al., 2015), post-translational hydroxylation (van Staalduinen and Jia, 2015) and the role of citrullination for the interaction between bacteria and human mucosal surfaces (Sofat et al., 2015). Several experimental papers report studies on post-translationally modified antimicrobial peptides known as lantibiotics. These are ribosomally synthesized peptides which can efficiently inhibit the growth of Gram-positive bacteria. A study by Zhou et al. (2015) described an engineering strategy to make the lantibiotics more effective in inhibiting several important bacterial pathogens. In another study, Khusainov et al. (2015) describe the active site of the nisin dehydratase. This enzyme, essential for the production of the lantibiotic nisin, converts serines, and threonines, to dehydroalanine and dehydrobutyrine residues, respectively. Interestingly, bacterial PTM systems, such as the N-glycosylation machinery, are also being exploited and engineered to facilitate the production of recombinant vaccines (Garcia-Quintanilla et al., 2014). Another focal point of the topic are the PTMs targeting the PII proteins, which are the key signal transduction proteins involved in the control of nitrogen metabolism in bacteria and archaea (Radchenko et al., 2014; Merrick, 2015). Finally, Stannek et al. (2015) contributed a study on a regulatory mechanism involving arginine phosphorylation and regulated proteolysis. In conclusion, the contributions in this topic reflect the diversity of bacterial PTMs. Several studies highlight one important emergent feature of PTM systems: their capacity to interact with each other, creating an additional level of complexity in the cellular regulation. This is one of the key features of bacterial PTM systems and a challenge which future studies will have to address.

Online recovery of nisin during fermentation coupling with foam fractionation

Online foam fractionation was proposed for recovering nisin during the fermentation of Lactococcus lactis subsp. Lactis ATCC 11454. Two strategies of fermentation coupling with foam fractionation were designed. In each strategy, sterile air and nitrogen were individually bubbled into the broth. The results showed that the activity of nisin in both continued to increase after the completion of the foam fractionation process. The maximum total nisin activity (4657IU/mL) was achieved when online foam fractionation was carried out using sterile air of 30mL/min between 6h and 12h, which was improved by 36.2% compared with that of the traditional batch fermentation (3420IU/mL) without online separation. This improvement is mainly attributed to the relief of product inhibition through online recovery of nisin, which demonstrates a promising process of industrial nisin production.

Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system.

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed-batch fermentation system with high fidelity (R(2) 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L(-1) h(-1) , 3 μg mL(-1) and 40%, respectively. While 1711 IU mL(-1) nisin was produced by L. lactis N8 in control fed-batch fermentation, 5410 IU mL(-1) nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed-batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed-batch fermentation.

Complete genome sequence of Lactococcus lactis S0, an efficient producer of nisin

Lactococcus lactis S0 is a nisin Z-producing strain isolated from milk, and the nisin production of the strain can reach 4000IU/ml under fermenting condition. Here, we present the complete genome sequence of L. lactis S0 which includes a single circular chromosome.

Use of Nisin-Producing Starter Cultures of Lactococcus lactis subsp. lactis on Cereal Based-Matrix to Optimize Preservative Factors over Fish Fermentation at 30°C Typical to Senegal

This study describes an application of bacteriocinogenic lactic acid bacteria to improve preservative factors over fish fermentation based on the most frequently used technique in Senegal. Traditional fish fermentation performed by immersion in salted sea water and incubation at 30°C for 24 to 48h to develop flavor, is characterized by a pH value around 7. The process led to the growth of Enterobacteriaceae to reach 9 log CFU/g. Four bacteriocinogenic lactic acid bacteria characterized in previous works were screened for decarboxylation activities. Only cultures of two Lactoccus lactis subsp. lactis (CWBI-B1410 and CWBI-B1426) producing nisin, a heat stable antibacterial peptide, did not produce histamine and tyrosine regarded as undesirable compounds on seafood products. The two lactococcal starter cultures grew well on a substrate based on millet, a local cereal. The AU/ml minimum inhibitory concentration of the antibacterial neutralized supernatant of the bacteriocinogenic starter cultures against Listeria monocytogenes was lower to those of many food poisoning or spoilage bacteria. Then, pieces of lean (Podamassys jubelini) and fat (Arius heudelottii) fish inoculated with 104-5 CFU/g of Listeriamonocytogenes, were immersed in the millet flour based substrate seeded with 105-6 CFU/g pure cultures of the nisinogenic bacteria to evaluate the control of the fermentation at 30°C. Listeria monocytogenes contaminated fish samples immersed in the millet-based substrate without starter cultures addition were used as negative controls. The starter culture fermentation gave the lowest fish pH reading. The pH was dropped and maintained to a final level lower to the minimum value for the growth of many Enterobacteriaceae genera reported in spoiled fish. The growth of Listeria monocytogenes was significantly inhibited by starter cultures compared to that in the negative controls. These results indicate that cultures of the two nisin producers on the new formulated matrix can be used to improve fish fermentation in Senegal.

Acetobacter pasteurianus DSM 3509 produces cobalamin

Seed coat is an important tissue for the regulation of imbibition and maintenance of the integrity of seed, and it is also the first seed barrier encountered by pests and pathogens. Seed cotyledons contain an array of proteins that may be involved in the protection of quiescent seeds against fungi. Now we know that seed coat from Theobroma cacao L. seeds contains an antifungal activity. In the present study inhibition tests of seed coat extracts against microorganisms isolated from cocoa bean fermentations were performed. Seed coat was extracted using surfacesterilization and filter-sterilization. To determine antifungal activity in seed coat extract agar diffusion and broth microdilution tests were used. Seed coat extract can inhibit growth of fungi (e.g. Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, Mortierella isabellina, Penicillium citrinum, Penicillium purpurogenum, Penicillium roquefortii). and yeasts (e.g. Candida krusei, Candida lipolytica, Candida guilliermondii, Cryptococcus laurentii, Rhodotorula mucilaginosa, Rhodotorula rubra, Saccharomyces cerevisiae, Schizosaccharomyces pombe). No inhibition effect could be detected against gram positive bacteria (e.g. Bacillus subtilis, Staphylococcus aureus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, Propionibacterium freudenreichii) and gram negative bacteria (e.g. Escherichia coli, Acetobacter pasteurianus, Acetobacter tropicalis, Acetobacter pomorum, Gluconobacter frateurii, Acetobacter orientalis). The minimum inhibitory concentration (MIC) of seed coat extract was determined. 25 mg/mL of seed coat extract can inhibit growth of fungi (e.g. Penicillium citrinum, Aspergillus niger, Penicillium purpurogenum), and 10 mg/mL seed coad extract can inhibit growth of yeasts (e.g. Saccharomyces cerevisiae, Rhodotorula rubra, Candida lipolytica). Nisin production in tofu by fermentation Nicole Illas, Raphael Cziskus, Caterina Hunniger, Agata Segieda, Xuan Zhu, Markus Fischer, Bernward Bisping University of Hamburg, Institute of Food Chemistry, Division of Food Microbiology and Biotechnology, Biocenter Klein Flottbek, Ohnhorststr. 18, D-22609 Hamburg, Germany Division of Food Chemistry, Bundesstr. 45, D-20146 Hamburg, Germany Tofu has a high nutritional content but on the other hand it can easily be spoiled. In Indonesia tofu production takes place mostly in small factories without an opportunity for pasteurization. Nisin production in tofu by fermentation with Lactococcus lactis ssp. lactis DSM 20729 may be a possibility to preserve tofu. The fermentation of tofu cubes (2*2 cm) submerged in water was performed for two days. Two methods of detection of nisin in fermented tofu were tested and compared by using inhibition test (modified method of Pongtharangkul and Demirci 2004), and a liquid chromatography electrospray ionization tandem mass spectrometry method (LC-ESI-MS/MS), based on ISO/TS 27106:2009. The detection limit was significantly lower by using the inhibition test. The nisin content was 0.19 mg/kg in tofu and 0.06 mg/L in liquid substrate. In comparison to this the detection limit for nisin using LC-ESI-MS/MS was 0.34 mg/kg in tofu. Matrix calibration of the liquid substrate could not be carried out by LC-ESI-MS/MS, because the background noise of the matrix was too high. Furthermore the production and distribution of nisin in tofu cubes was investigated. The nisin concentration on the surface of tofu was 2.64 mg/kg and so it was nine times higher compared to the interior of tofu. The optimum storage condition for fermented tofu was a combination of low temperature and low pH-value. An alternative to preserve tofu may be fermentation of tofu with L. latis ssp. lactis. Nisin concentration of complete tofu cubes could be successfully determined by using both methods. The inhibition test proved to be more sensitive for nisin detection in tofu. Pongtharangkul T, Demirci A (2004) Evaluation of agar diffusion bioassay for nisin quantification. Appl Microbiol Biotechnol 65: 268-272 International Organization for Standardization: ISO/TS 27106 (2009) Cheesedetermination of nisin A content by LC-MS and LC-MS/MS. Geneva, Switzerland

Fermentative Nisin Production in Tofu for its Preservation

Fermentative Nisin Production in Tofu for its Preservation

Structure of the nisin leader peptidase NisP revealing a C-terminal autocleavage activity.

Nisin is a widely used antibacterial lantibiotic polypeptide produced by Lactococcus lactis. NisP belongs to the subtilase family and functions in the last step of nisin maturation as the leader-peptide peptidase. Deletion of the nisP gene in LAC71 results in the production of a non-active precursor peptide with the leader peptide unremoved. Here, the 1.1 Å resolution crystal structure of NisP is reported. The structure shows similarity to other subtilases, which can bind varying numbers of Ca atoms. However, no calcium was found in this NisP structure, and the predicted calcium-chelating residues were placed so as to not allow NisP to bind a calcium ion in this conformation. Interestingly, a short peptide corresponding to its own 635-647 sequence was found to bind to the active site of NisP. Biochemical assays and native mass-spectrometric analysis confirmed that NisP possesses an auto-cleavage site between residues Arg647 and Ser648. Further, it was shown that NisP mutated at the auto-cleavage site (R647P/S648P) had full catalytic activity for nisin leader-peptide cleavage, although the C-terminal region of NisP was no longer cleaved. Expressing this mutant in L. lactis LAC71 did not affect the production of nisin but did decrease the proliferation rate of the bacteria, suggesting the biological significance of the C-terminal auto-cleavage of NisP.

Modeling the batch bacteriocin production system by lactic acid bacteria by using modified three-dimensional Lotka–Volterra equations

Different batch cultures of Lactococcus lactis CECT 539, a nisin-producing strain, were carried out in culture media prepared with whey and mussel processing wastes. From these cultures, a reasonable system of differential equations, similar to the three-dimensional Lotka–Volterra two predators-one prey model, was set up to describe, for the first time, the relationship between the absolute rates of growth, pH drop and nisin production.Thus, the nisin production system was described as a three-species (pH, biomass and nisin) ecosystem. In this case, both nisin and biomass production were considered as two pH-dependent species that compete for the nitrogen source. Excellent agreement (R2 values ≥0.9885) resulted between model predictions and the experimental data, and significant values for all the model parameters were obtained. The developed model was demonstrated (R2 values ≥0.9874) for five batch cultivations of the strains L. lactis CECT 539 in MRS broth and Lactobacillus sakei LB 706 (sakacin A producer), Pediococcus acidilactici LB42-923 (pediocin AcH producer), L. lactis ATCC 11454 (nisin producer) and Leuconostoc carnosum Lm1 (leuconocin Lcm1 producer) in TGE broth. These results suggest that the batch bacteriocin production system in these culture media can be successfully described by using the Lotka–Volterra approach.

Diversity Analysis, Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)5-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.

Genome shuffling of Lactococcus lactis subspecies lactis YF11 for improving nisin Z production and comparative analysis

Nisin has been widely used in the food industry as a safe and natural preservative to increase the shelf time of many foods. In this study, genome shuffling was applied to improve nisin Z production of Lactococcus lactis ssp. lactis YF11 (YF11) via recursive protoplast fusion. Ultraviolet irradiation and diethyl sulfate mutagenesis were used to generate parental strains for genome shuffling. After 4 rounds of genome shuffling, the best-performing strain F44 was obtained, which showed dramatic improvements in tolerance to both glucose (ranging from 8 to 15% (wt/vol) and nisin (ranging from 5,000 to 14,000IU/mL). Fed-batch fermentation showed that the nisin titer of F44 was up to 4,023IU/mL, which was 2.4 times that of the starting strain YF11. Field emission scanning electron microscope micrographs of YF11 and F44 revealed the apparent differences in cell morphology. Whereas YF11 displayed long and thin cell morphology, F44 cells were short and thick and with a raised surface in the middle of the cell. With the increasing glucose and nisin content in the medium, cells of both YF11 and F44 tended to become shrunken; however, alterations in YF11 cells were more pronounced than those of F44 cells, especially when cultured in tolerance medium containing both nisin and glucose. Nuclear magnetic resonance analysis demonstrated that the structure of nisin from YF11 and F44 was the same. Expression profiling of nisin synthesis related genes by real-time quantitative PCR showed that the transcription level of nisin structural gene nisZ and immunity gene nisI of F44 was 48 and 130% higher than that of the starting strain YF11, respectively. These results could provide valuable insights into the molecular basis underlying the nisin overproduction mechanism in L. lactis, thus facilitating the future construction of industrial strains for nisin production.

Nisin production in a chitin-including continuous fermentation system with Lactococcus lactis displaying a cell wall chitin-binding domain

The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1-0.9 h⁻¹) and initial glucose concentrations (20-60 g l⁻¹). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h⁻¹ dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml⁻¹ and 9,450 IU ml⁻¹ h⁻¹, respectively) with the combination of a 0.9-h⁻¹ dilution rate and a 40-g l⁻¹ initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.

Study on the process of fermentation coupling with foam fractionation and membrane module for nisin production

ABSTRACTNisin, an antimicrobial peptide, is commercially used as a food preservative and a promising alternative in medical applications for inhibiting bacterial infection in both humans and livestock. It is produced by fermentation using certain strains of Lactococcus lactis ssp. lactis. In traditional fermentation, the yield of nisin has always been limited by adsorption of nisin onto its producer, enzymatic degradation and product inhibition. This limitation can be solved by fast removal of the product from the broth during fermentation. In the current work, a technology of fermentation coupling with foam fractionation and membrane module was developed to achieve in situ removal of nisin produced by L. lactis ssp. lactis ATCC 11454 from the broth. The effects of air flow rate, the initial aeration time and interval time on the performance of the technology were studied to increase the yield of nisin. The results showed that under the optimal conditions of air flow rate 60 mL/min, the initial aeration time of the fourth hour and regular 30‐min interval, the total nisin titer reached 3946 IU/mL, increased by 24.2%, compared with the control group (3177 IU/mL). Therefore the coupling technology can effectively weaken the feedback inhibition and thus increase the yield of nisin by in situ fast removal of the product from broth during fermentation. © 2014 Curtin University of Technology and John Wiley & Sons, Ltd.

Non-culturability and Nisin Production of Lactococcus lactis

We studied formation of non-culturable forms of three bacteriocin (nisin) producing strains of Lactococcus lactis sub sp. lactis: MSU, 729 and F-116 under carbohydrate starvation stress. Two different types of inoculum were applied: A) unwashed cells with culture liquid, B) cells washed twice with normal 0,9%saline. Resulting total numbers of cells were 0.6 1.0×108 cells/ml for both types of inoculum. Population obtained using type A inoculum demonstrated active growth phase within first 1-5 days of incubation (up to 2.4×109 cells/ml) while those obtained using type B inoculum did not grow within that period. Type B population of strain MSU showed phenotypic dissociation that resulted in appearance of micro colonies. After that, we observed active growth phase (up to 5.2×109 cells/ml). Type B cultures of strains 729 and F-116 did not grow during the whole experiment. It was shown that type A population shifted into non-culturability faster than type B. This is due to differences in metabolic strategies and stress sensitivity of these types of population. After 1 year of incubation (383 days) culturability decreased by 3 orders of magnitude for type B (5 orders for type B population of strain MSU) and by 6 orders of magnitude for type A population. We also observed considerable reduction of cell size for type A population of strains 729 and F-116. Studies of bacteriocin activity showed that in type B population cells were up to 78 times more productive compared to those of type A cultures. This phenomenon can be explained by differences in survival strategies of population that use antibacterial potential of bacteriocins for their benefit.

Pediococcus acidilacticiUL5 andLactococcus lactisATCC 11454 are able to survive and express their bacteriocin genes under simulated gastrointestinal conditions

The aim of this work is to study the expression of stress genes and those involved in pediocin and nisin production in Pediococcus acidilactici UL5 and Lactococcus lactis ATCC11454 under simulated gastrointestinal (GI) physiological conditions. The two strains were fed to a dynamic GI model (TIM-1). Samples were taken from different compartments and analysed for strain survival as well as for the expression of pediocin PA-1 operon, nisin A production gene and stress genes using RT-qPCR. Ileal-delivered efflux showed a survival rate of 17 and 0·0007% for Ped.acidilactici and La.lactis, respectively. Pediocin operon genes from stressed cells were generally expressed at least at the same level as for unstressed cells. However, pedA is up-regulated in the effluent at 120 and 180min. Nisin A genes were always up-regulated with particularly in the stomach after 70min compared with control. Bacteriocin production of Ped.acidilactici UL5 and Lc.lactis ATCC 11454 are not affected by upper GI simulated conditions and thus could be considered as relevant probiotic candidates. This study demonstrates the capacity of lactic acid bacteria to survive and express their bacteriocins genes under simulated GI conditions.

Effect of Bile on Nisin-Mediated Antibacterial Activity and the Expression of Nisin Genes of Lactococcus lactis W8

The capability of Lactococcus lactis to produce nisin in the presence of bile in the intestinal environment remains an intriguing question. The aim of this study was to determine the effects of bile on production of nisin and the mRNA expression of nisin genes of L. lactis W8. The strain L. lactis W8 was grown on glucose in the absence and presence of bile (0.005-0.08 %) and the antibacterial activities of culture supernatants were determined. In culture with 0.035 % bile, the nisin activity was significantly reduced (400 AU/mL) within 5 h compared to that in the control without bile (2000 AU/mL), while growth of the cells was only slightly affected. In the presence of 0.07 % bile no nisin activity of the strain was manifested. Consistent with these results, mRNA expression of nisin-biosynthetic genes nisZ, nisRK, nisI, and nisF was down-regulated by 7.5-, 2.5-, 1.7-, and 6.0-fold, respectively in cells grown in the presence of bile (0.07 %) as compared to control culture without bile. The present study suggested that bile inhibited transcription of nisin genes. Nisin-production in intestine by orally administered L. lactis, thus, does not occur since complete inhibition of nisin-production by bile is observed at a concentration much lower than the physiological concentration (0.3 %) of bile present in the human intestine. The molecular mechanism underlying the bile-mediated inhibition of nisin genes remains to be elucidated. This is the first report on bile-mediated inhibition of nisin genes.

Isolation of bacteriocinogenic strain of Lactococcus lactis subsp. lactis from rocket salad (Eruca sativa Mill.) and evidences of production of a variant of nisin with modification in the leader-peptide

In this study we have isolated and identified strains of lactic acid bacteria (LAB) that are potential bacteriocin producers from rocket salad samples acquired in Sao Paulo, SP, Brazil. Using the PCR-ARDRA method, Lactococcus lactis subsp. lactis MK02R was identified and characterized as the only bacteriocinogenic among 12 strains initially isolated. The analysis of the antimicrobial peptide produced by MK02R revealed the presence of nisin variant, a well-known lantibiotic, and one of the most studied and important antimicrobial peptides known today. The bacteriocin MK02R showed heat stability for 1 h at 60 °C and 100 °C and it was inactivated by treatment with proteolytic enzymes. The antimicrobial activity did not change with pH adjustment between 2.0 and 9.0 and the production was stimulated by the addition of 0.5 and 1.0% cysteine in MRS broth at 37 °C. Bacteriocin MK02R inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Lactobacillus delbrueckii, Lb. sakei subsp. sakei, Listeria innocua and Listeria monocytogenes from different serological groups. Low levels of adsorption of bacteriocin MK02R were detected in the cell surface of the producer cells suggesting that bacteriocin MK02R remains bound to the outer surface and that it is released when the pH of the environment increases. The chromatographic studies and the genetic tests using primers nisF and nisR related to specific genes of nisin production confirmed that the antimicrobial MK02R is a natural variant of nisin. The partial sequencing of the reconstructed protein showed that the peptide has an amino acid change in the sequence of the leader peptide compared with nisin A, Z, Q, U and F, but the structure of the mature is homologous to that of nisin F.

Immobilization of nisin producer Lactococcus lactis strains to chitin with surface-displayed chitin-binding domain

In this study, nisin producer Lactococcus lactis strains displaying cell surface chitin-binding domain (ChBD) and capable of immobilizing to chitin flakes were constructed. To obtain ChBD-based cell immobilization, Usp45 signal sequence with ChBD of chitinase A1 enzyme from Bacillus circulans was fused with different lengths of PrtP (153, 344, and 800 aa) or AcmA (242 aa) anchors derived from L. lactis. According to the whole cell ELISA analysis, ChBD was successfully expressed on the surface of L. lactis cells. Scanning electron microscope observations supported the conclusion of the binding analysis that L. lactis cells expressing the ChBD with long PrtP anchor (800 aa) did bind to chitin surfaces more efficiently than cells with the other ChBD anchors. The attained binding affinity of nisin producers for chitin flakes retained them in the fermentation during medium changes and enabled storage for sequential productions. Initial nisin production was stably maintained with many cycles. These results demonstrate that an efficient immobilization of L. lactis cells to chitin is possible for industrial scale repeated cycle or continuous nisin fermentation.

Effect of Cameline Nisin isolated from Lactococcus lactis sub sp. lactis on<br>Staphylococcus aureus sp.

The camel milk differs from milk of other species by the presence of a powerful protector system, linked to relatively high levels of lysozyme, lactoperoxidase, lactoferrin, component-3 of proteose peptone (PP3), organic acid, hydrogen peroxide and bacteriocins produced by lactic acid bacteria. The study aims to estimate the part of a bacteriocin (type nisin) produced by a strain of Lactococcus lactis sub sp lactis isolated from camel milk, in this protective system and its antibacterial activity against a strain of Staphylococcus aureus isolated from a bovine milk sample. Optimize conditions for a nisin production, by the producing strain isolated and purified, was carried out, by glucose addition (1%) to the M17 medium. Antagonism tests by the disk method showed inhibition zones (IZ O ) from 6 to 8mm in diameter depending on the incubation time of the producing strain. Thus, for incubation periods of 18h, the production of camel nisin appeared to be maximum (IZ= O 8mm) with a minimum growth of Lactococcus lactis sub sp lactis (pH 6, 37). Optimum growth of camel Lactococcus lactis sub sp lactis strain (pH 6.17) and an optimum production of nisin (IZ= O 8mm) were registered for a 24 hours incubation period.